抗脓毒症天然活性先导化合物地骨皮乙素的结构优化研究

Sepsis is triggered by numerous pathogen-associated molecular patterns (PAMP). The current drugs can only neutralize one of PAMP, LPS, therefore they are inefficient in clinical practice. Recently the multi-target anti-PAMP strategy has been applied in researches. Kukoamine B (KB), a natural product from Traditional Chinese Medicine, has been found to neutralize both LPS and bacterial DNA, and the mechanisms for the interaction between KB and LPS/bacterial DNA have been elucidated in our studies. We also found that KB can neutralize peptidoglycan and lipteichoic acid, and the activities have yet to be improved. The amino and hydroxyl groups are important elements of the pharmacophore, and the hydrogen and hydrophobic bonding are the major forces. Based on these findings, a series of KB analogues with diverse structures and binding abilities will be synthesized according to the hydrogen bonding, hydrophilic/hydrophobic properties and steric hindrance according to the binding modes of KB to the targets the current study. Following the analysis of the quantitative relationships between the categories, numbers and positions, as well as the size, of the functional groups of KB, the structure of KB will be optimized to generate more effective compounds. These studies will provide theoretical foundation for the rational design of drugs for the treatment of sepsis and more candidates for the drug discovery that targets PAMP.

脓毒症是多种病原体相关分子模式(PAMP)共同作用的结果，但现有药物仅能针对其中一种PAMP，即脂多糖(LPS)，临床疗效并不理想。近年来，多靶点拮抗PAMP的策略得到广泛认可和应用。我们首次发现中药成分地骨皮乙素(KB)可同时拮抗LPS和细菌DNA两种PAMP，并阐明了KB与其相互作用的机理。我们还发现，KB对另外两种PAMP即肽聚糖和脂磷壁酸也有作用，但活性尚有待提升。氨基和羟基是KB重要的药效结构元素，氢键和疏水作用是主要作用力。基于上述发现，本研究拟在KB与靶点结合机理的分析基础上，针对氢键、亲/疏水性及空间位阻等特性，设计结构多样、靶点亲和力与活性强弱各不相同的KB衍生物，定量分析其官能团种类、数目、位置和大小等结构特征与活性间的关系，进而指导结构优化，获得新的更为有效的多靶点化合物，为探索多靶点抗脓毒症药物合理设计理论提供有益实践，也为靶向PAMP的药物开发提供更好的候选分子。

脓毒症是严重感染患者常见的致命性并发症。病原体相关分子模式（PAMPs）是治疗脓毒症药物研究的重要靶点，我们从中药地骨皮中发现了同时拮抗脂多糖（LPS）和细菌基因组DNA（CpG DNA）两种重要模式分子的先导物地骨皮乙素，但其如何作用于上述两种靶标尚不完全清楚。为阐明该科学问题，并进行结构优化以设计具有更好成药性的化合物，本项目采用分子动力学、热力学及计算机模拟技术研究了地骨皮乙素与靶点分子的结合机理，设计了多种类型的地骨皮乙素衍生物，结合虚拟筛选结果从中选择性合成了数个新化合物，并且通过体内外药理实验进行了活性验证。结果显示：（1）地骨皮乙素主要以氢键和疏水作用与LPS、CpG DNA和脂磷壁酸（LTA）结合。地骨皮乙素与LPS结合的主要位点位于LPS的磷酸官能团，同时也与其脂肪酸和Kdo侧链有结合作用；地骨皮乙素与CpG DNA结合的主要部位在于其GACGTT这一核心序列；地骨皮乙素与LTA结合的主要位点是LTA的磷酸基团和N-乙酰甘露糖胺部位；（2）设计了9个系列共计31个地骨皮乙素衍生物，通过虚拟筛选预测化合物H1对LPS和CpG DNA的活性更优；（3）采用生物传感器、鲎试剂检测以及ELISA检测技术，从不同角度验证H1对LPS和CpG DNA的活性最好。在RAW264.7细胞模型上，对LPS和CpG DNA诱导释放TNF-α的抑制作用分别比地骨皮乙素提高了1倍和5倍。在CLP模型动物上，H1显著提高动物存活率，具有统计学差异，并呈剂量-效应依赖关系。该研究对全面阐释地骨皮乙素的作用机制具有重要意义，也为进一步深入进行结构优化与合理药物设计，发现更好的多靶点抗脓毒症药物奠定了基础。